1. Field of the Invention
This invention relates to use of a method for screening nonpathogenic anti-cariogenic strains, and products and methods using such strains for treatment and prophylaxis of dental caries caused by oral bacteria such as Streptococcus mutans, and other caries-causing pathogens.
2. Description of the Related Art
The oral cavity of humans and other mammals contains many different species of bacteria, including a number of different species of Lactobacillus. Caries is a disease caused by bacteria. In 1890, Miller in “Chemico-Parasitic Theory” presented the hypothesis that caries is caused by oral bacteria producing acids from digestive carbohydrates, which dissolve the hydroxyhepatite of the teeth. It was later confirmed in gnotobiotic rats, for example, that normal oral bacterial flora, are involved, primarily of the mutans streptococci group, and also that the lactobacilli group is involved. These “acidogenic” species resident in the oral cavity are associated with the presence and onset of dental caries (Locsche W J, Microbiolog Rev.,1986:50:353-380). There are seven bacterial species within the group mutans streptococci, with Streptococci mutans (serotype c,e,f) being found in 90% of all human isolates (Linder L., Oral Mikrobiologi 1996, ISBN 91-7205-037-3). There is abundant evidence that the initiation of caries requires a relatively high proportion of S. mutans within dental plaque. These bacteria adhere well to the tooth surface, produce higher amounts of acid from sugars than other bacterial types, can survive better than other bacteria in an acid environment, and produce extracellular polysaccharides from sucrose. When the proportion of S. mutans in plaque is high (in the range of 2-10%), a patient is at high risk for caries. When the proportion is low (less than 0.1%), the patient is at low risk. Because they are more acid tolerant than other bacteria, acid conditions within plaque favor the survival and reproduction of mutans streptococci. Two other types of bacteria are also associated with the progression of caries through dentin. These are several species of Lactobacillus, and Actinomyces viscosus. These bacteria are also highly acidogenic and survive well in acid conditions. The involvement of Lactobacillus in dental caries has been established (Smith et al., Microbios 105: 77-85, 2001). In fact, estimation of the lactobacillus counts in saliva, in addition to the estimation of mutans streptococci counts, using different selective media or other techniques, has been used for many years as a “caries test” and to attempt to identify groups at high risk for caries. Thus, Lactobacillus strains, some isolated from human dental plaque, may be highly cariogenic (Fitzgerald et al., J. Dent. Res. 60: 919-926, 1981.
For a bacteria to be a primary pathogen in the formation of dental caries it is required that it have a combination of several of the required characteristics (Linder, 1996): ability to adhere and colonize on the teeth surface; ability to accumulate in large numbers on a limited surface of the teeth; ability to quickly produce acid from carbohydrates found in foods; and ability to continue acid production even under low pH in the dental plaque.
Dietary sucrose changes both the thickness and the chemical nature of plaque. Mutans streptococci and some other plaque bacteria use the monosaccharide components (glucose and fructose) and the energy of the disaccharide bond of sucrose to assemble extracellular polysaccharides. These increase the thickness of plaque substantially, and also change the chemical nature of its extracellular space from liquid to gel. The gel limits movement of some ions. Thick gel-plaque allows the development of an acid environment against the tooth surface, protected from salivary buffering. Plaque which has not had contact with sucrose is both thinner and better buffered. A diet with a high proportion of sucrose therefore increases caries risk. Thicker plaque occurs in pits and fissures and, in patients with poor oral hygiene, near the gingival margin.
Given this concept of the nature of the disease, it is clear that prevention and treatment of dental caries requires hindering the effects of S. mutans, for example, through dietary change as means of reducing the substrate for the bacteria, to reinforce the surface structure of the teeth or reduction of the number of S. mutans bacteria. Thus, treatments that have been tried include: efforts at changing the microflora, using agents such as topical chlorhexidine and topical fluoride; reducing the amount of dietary sucrose, by dietary change and substitution for sweeteners more difficult to metabolize by S. mutans, such as Sorbitol, Aspartan, Xylitol; decreasing the frequency of eating, by dietary choice; adding fluoride, particularly through daily application during tooth brushing; and increasing salivary flow, using mechanical stimulation during vigorous chewing to enhance flow, by changing drugs which reduce flow, or by using drugs to enhance flow. Different approaches has been evaluated for preventing dental caries, for example, one composition uses a lytic enzyme produced by a bacteriophage specific for Streptococcus mutans (U.S. Pat. No. 6,399,098 of Fischetti et al.). Also, a strain of Lactobacillus zeae has been modified through genetical engineering to produce an antibody on its surface to neutralize the detrimental streptococcal bacteria, (Hammarstrom L., July 2002 issue of Nature Biotechnology), however this approach with genetically modified organisms faces an unknown safety approval situation.
In addition, one strain of Lactobacillus rhamnosus (strain GG) has been promoted as a probiotic method of reducing Streptococcus sabrinus and mutans streptococci generally (Nase et al., Caries Res. 35: 412-420, 2001). Further work showed that use of this strain as a starter in fermenting milk did not influence the titer of antibodies against human cariogenic bacteria that were present in the milk (Wei et al., Oral Microbio. & Immunol. 17: 9-15, 2002. L. rhamnosus GG differs from L. reuteri in many ways, including fermentation characteristics and isolation source. Other microorganisms that have been found to have inhibitory activity against the formation of dental plaque include Enterococcus, Lactobacillus acidophilus V20, and Lactobacillus lactis 1370 (Oh, U.S. Pat. No. 6,036,952). In order to inhibit S. mutans, other work has been done using so called “competitive exclusion” concepts. For example, L. reuteri strain ATCC 55730 has been shown to inhibit S.mutans (Nikawa H. et al, News release by Hiroshima University Jul. 11, 2002). A tablet product which is on the market in Japan called LS1, containing a strain of Lactobacillus salivarius (LS 1) (by Frente Ltd. Japan) is claimed to inhibit S.mutans. 
Strains of a wide variety of Lactobacillus species, including Lactobacillus reuteri, have been used in probiotic formulations. Lactobacillus reuteri is one of the naturally occurring inhabitants of the gastrointestinal tract of animals, and is routinely found in the intestines, and occasionally in the birth channel, breast milk and mouth of healthy animals, including humans. It is known to have antibacterial activity. See, for example, U.S. Pat. Nos. 5,439,678, 5,458,875, 5,534,253, 5,837,238, and 5,849,289. When L. reuteri cells are grown under anaerobic conditions in the presence of glycerol, they produce the antimicrobial substance known as reuterin (β-hydroxy-propionaldehyde). Other antimicrobial substances beside the traditional organic acids have also been reported such as “Reutericyclin” (Höltzel, A. et al. Angewandte Chemie International Edition 39, 2766-2768, 2000) and “PCA (pyroglutamic acid)” (Yang, Z. Dissertation, Univ. of Helsinki, March 2000). Lactobacilli, including L. reuteri, are also well known to have the ability to inhibit other organisms such as S. mutans through local competition of nutrients and other metabolic interactions. Immunomodulating and anti-inflammatory activity has also been associated with L. reuteri. Mucin binding proteins of L.reuteri have been isolated and described. See, for example, U.S. Pat. No. 6,100,388.
Lactobacillus strains have been reported to adhere to various cell lines and host mucus. This has been speculated to be important for probiotic activity and is derived from the concept of virulence factors in pathogenic bacteria, where vast arrays of such interactions have been discovered during the last decades (Klemm, P. and Schembri, M. A. (2000) Bacterial adhesins: function and structure. Int. J. Med. Microbiol. 290, 27-35.)
While the possibility of effective antibacterial activity by L. reuteri is known, and certain binding characteristics of L.reuteri such as mucin binding are known, and S.mutans inhibiting effects of L.reuteri strain ATCC 55730 and Lactobacillus GG ATCC 53103 are also known, it was not previously known that substantial differences existed between lactobacilli strains in their ability to reduce the number of Streptococci mutans in the oral cavity and thereby caries, as a consequence of both inhibiting effect and binding activity, nor that such strains could be selected.
It is therefore an object of the invention to provide better strains of Lactobacillus which have been selected for their capability of reduce the number of S. mutans in the mouth through antimicrobial activity in combination with good binding to the oral mucins and dental plaque and thereby prevent, reduce or treat dental caries. It is a further object of the invention to provide products containing said strains, including agents for prophylaxis or treatment of caries associated with S.mutans for administration to humans.
Other objects and advantages will be more fully apparent from the following disclosure and appended claims.